A dual MMP9/MMP14 Axis Regulates Osteoclast Bone Resorptive Function Abstract Excessive osteoclast (OC) activity is responsible for a wide range of bone diseases, ranging from osteoporosis and rheumatoid arthritis to tumor-induced osteolysis. Despite the development of several anti-resorptive therapeutics, their palliative effects are often limited or accompanied by unwanted side effects. In this regard, cathepsin K has been targeted for therapeutic intervention given its key role as a collagenolytic enzyme capable of degrading type I collagen, the dominant protein component of the bone extracellular matrix. Interestingly, however, OCs also express matrix metalloproteinases whose function in OC bone-resorptive activity remains largely undefined. Herein, we utilized unbiased transcriptome screening as wells as ex vivo assays to identify MMP9 and MMP14 as the two dominant MMPs expressed by OCs. However, after generating Mmp9-/- or myeloid-specific Csf1r-Cre/Mmp14f/f OCs, preliminary studies suggest that neither proteinase plays a key role in bone resorption. Unexpectedly, we find that Csf1r-Cre/Mmp14f/f/Mmp9-/- double knockout OCs display major defects in bone resorption in vitro and in vivo. In an effort to define the mechanisms underlying a combined requirement for MMP9 and MMP14 in OC function, preliminary analysis of wild-type versus double knockout OC gene expression identified unexpected alterations in carbohydrate metabolism, zinc finger binding and mitochondrion. These findings led us to posit and confirm that working together, MMP9 and MMP14 play a critical role in regulating a zinc finger transcription repressor Zeb1-arbitrated metabolic pathway to govern OC activation in tandem with the ability of the MMPs to mediate bone collagenolytic effects. Thus, we propose to i) characterize the cooperative role of MMP9/MMP14 in controlling osteoclast function and bone resorption in vitro and in vivo, utilizing both myeloid- and OC-specific conditional knockout mice, ii) identify a novel MMP9/MMP14-Zeb1 axis in regulating energy metabolism and osteoclast activity in vitro and in vivo, and iii) Define the dual roles of the MMP9/MMP14 co-dependent proteolysis of galectin-3 membrane lattice as upstream of Zeb1, in parallel with bone type I collagenolysis in regulating osteoclast bone-resorptive function. The unique proteolytic and metabolic signaling route outlined in this proposal should provide new insights into OC-mediated bone remodeling, and advance the search for improved therapeutic strategies designed to prevent pathologic bone loss.